ABSTRACT Pathways regulating cell fate decisions during the earliest stages of embryo development are complex and remain poorly understood. Most developmental studies concentrate on transcriptional regulation, and thus fail to take into account the fact the steady-state levels of RNAs are dictated as much by their rate of decay as by their rate of synthesis. This proposal focuses on Nonsense-Mediated RNA Decay (NMD), a highly conserved RNA degradation pathway that selectively degrades specific subsets of transcripts. Loss or knockdown of NMD factors leads to developmental defects in species spanning the phylogenetic scale, including humans. One of the most striking findings is that loss of all but one of the examined NMD factors leads to early embryonic lethality in mice, as well as other higher eukaryotes. To date, there have been no studies examining the underlying mechanism. This application addresses this question using mouse models and normal human cell lines, coupled with genome-wide molecular approaches at the single-cell level. The overarching hypothesis is that NMD degrades specific transcripts to control developmental decisions in the early embryo. Support for this hypothesis comes from previously published studies on neural differentiation from the Wilkinson laboratory, in which they identified NMD-based circuits that maintain the neural stem cell state. Neural differentiation signals trigger the expression of microRNAs that repress NMD, leading to stabilization of pro-neural transcripts and the consequent initiation of neural differentiation. This work led to the hypothesis that NMD also promotes the undifferentiated state in other cell lineages. In support, the Wilkinson laboratory demonstrated that NMD magnitude tends to be high in undifferentiated cells and is downregulated upon differentiation. Furthermore, they showed that NMD magnitude plays a specific role in regulating cell fate decisions of primary germ layers in human embryonic stem cells (hESCs), such that NMD downregulation drives endoderm differentiation, while NMD upregulation augments mesoderm differentiation. In addition, the Wilkinson laboratory recently discovered a factor that likely plays a critical role in controlling NMD magnitude in hESCs and the early embryo. This factor, UPF3A, was previously regarded as a weak NMD factor from artificial tethering experiments, but we recently showed that it acts primarily as a potent NMD repressor through extensive loss- and gain-of-function experiments, both in vitro and in vivo. This suggests the hypothesis that UPF3A serves as a molecular rheostat, thereby controlling differentiation events critical in early embryonic development. In support of this hypothesis, the Wilkinson laboratory showed that loss of UPF3A causes early embryonic lethality, accompanied by morphological defects at the pre-implantation stage. The goals of my proposal are to (i) dissect the specific roles of NMD in early pre-implantation embryo development, (ii) elucidate the role of a NMD repressor identified by the Wilkinson laboratory?UPF3A?in embryogenesis, and (iii) define molecular circuits by which NMD acts in early development using hESCs to model human development.